改进型颈动脉注射法构建裸鼠脑转移瘤模型

Establishment of Brain Metastasis Model in Nude Mice by Modified Carotid Artery Injection Method

关键词:  肝癌  脑转移  颈内动脉注射  动物模型  活体成像

Key words:  liver cancer  brain metastasis  internal carotid artery injection  animal model  in vivo imaging

摘要:

目的 建立桡足类超绿色荧光蛋白（copepod super green fluorescent protein，copGFP）与纳米荧光素酶（nano luciferase，Nluc）标记的MHCC97-H人肝癌细胞株，通过改进型颈动脉注射法构建应用小动物活体成像系统观察的裸鼠脑转移模型。方法 使用携带目的基因的慢病毒载体质粒通过磷酸钙法转染293FT细胞，包装慢病毒后感染MHCC97-H细胞，经嘌呤霉素筛选感染阳性细胞，通过有限稀释法纯化得到稳定表达copGFP及Nluc的单克隆细胞株。通过改进型颈动脉注射法构建裸鼠脑转移模型，应用活体成像系统监测颅内转移灶生长情况。结果 成功建立了copGFP与Nluc标记的MHCC97-H肝癌细胞稳定株。分别使用稳定株与原始株细胞通过改进型颈动脉注射法造模，2组荷瘤裸鼠体质量变化及生存曲线无统计学差异，使用稳定株造模裸鼠可通过活体成像系统实时监测到脑转移的形成，所检测生物发光信号强度随肿瘤生长逐渐增强。结论 经copGFP与Nluc标记的MHCC97-H人肝癌细胞通过改进型颈动脉注射法成功构建裸鼠脑转移模型，可通过活体成像系统进行无创、连续可视化监测，为肝癌脑转移机制的研究及治疗药物的研发提供理想的动物模型。

Abstract:

OBJECTIVE To establish copepod super green fluorescent protein(copGFP) and nano luciferase(Nluc) labeled MHCC97-H human hepatoma cells. To construct a model of brain metastasis in nude mice, in which can be observed by in vivo imaging system. METHODS The lentiviral vector plasmid carrying the target gene was used to transfect 293FT cells by calcium phosphate method, and the lentivirus was packaged to infect MHCC97-H human hepatoma cells. The positive cells were screened by puromycin, and the monoclonal cell strain stably expressing copGFP and Nluc were purified by limiting dilution assay. The brain metastasis model of nude mice was established by modified carotid artery injection, and the growth of intracranial metastasis was monitored by in vivo imaging system. RESULTS Successfully established a stable strain of MHCC97-H human hepatoma cells which were labeled by copGFP and Nluc. The stable strain and the original strain were used to establish the model by modified carotid artery injection method, and there was no statistically significant difference in body mass change and survival curve between the two groups of tumor-bearing nude mice. The formation of brain metastasis in nude mice with stable strain could be monitored in real time by in vivo imaging system, and the intensity of bioluminescence signal was gradually enhanced with the growth of tumor. CONCLUSION MHCC97-H human hepatoma cells labeled by copGFP and Nluc are successfully established and triumphally constructed a model of brain metastasis in nude mice by a modified carotid artery injection method. This experimental model can be non-invasively and continuously visually monitored by in vivo imaging system, which provides an ideal murine model for the study of the brain metastasis mechanism of liver cancer and the development of therapeutic drugs.